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Article

Absolute Quantitation of Proteins by a Combination of Acid Hydrolysis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Olga A. Mirgorodskaya,^† Roman Körner,*^‡ Alexander Novikov,^† and Peter Roepstorff^§

Department of Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark, and Institute of Cytology of the Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia

Anal. Chem., 2004, 76 (13), pp 3569–3575

DOI: 10.1021/ac035389y

Publication Date (Web): May 15, 2004

†

Institute of Cytology of the Russian Academy of Sciences.

Corresponding author. Fax: +49 89 8578 3102. E-mail: rkoerner@ biochem.mpg.de.

‡

Max-Planck Institute of Biochemistry, Department of Cell Biology, Am Klopferspitz 18a, D-82152 Martinsried, Germany.

University of Southern Denmark.

Abstract

Quantitation by mass spectrometry is increasingly used to monitor protein levels in biological samples. Most of the current methods are based on the relative comparison of protein quantities but are not suited for the determination of the absolute amount of a given protein. Here we describe a method for the absolute quantitation of proteins that is based on amino acid analysis by matrix-assisted laser desorption/ionization mass spectrometry. Proteins are completely hydrolyzed by acid hydrolysis and then mixed with standards of isotopically labeled amino acids. For the presented study, lysine, leucine, and two different types of labeled arginine were examined as standards. Quantitation of proteins is then achieved by measuring the ratios of labeled and unlabeled amino acids. The method has a sensitivity down to the low-femtomole range and can be applied to quantitate proteins separated by gel electrophoresis. Furthermore, we demonstrate that a mixture of two proteins can be quantitated using two labeled amino acids simultaneously.

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